Glycophosphoceramides from Plants

Transcription

1 THE JOURNAL OF BOLOGCAL CHEMSTRY Vol. 256, No. 5, ssue of August, pp , 98 Prcnted m U.S.A. Glycophosphocermides from Plnts PURFCATON AND CHARACTERZATON OF A NOVEL TETRASACCHARDE DERVED FROM TOBACCO LEAF GLYCOLPDS* (Received for publiction, Mrch 25, 98) Thoms C.-Y. HsiehS, Robert L. Lestergl, nd Roger A. LinegJ From the Deprtment of Biochemistry, University of Kentucky College of Medicine, Lexington, Kentucky 4536 A glycophosphocermide concentrte prepred from tobcco leves ws shown to contin mixture of relted lipids (Kul, K., nd Lester, R.L. (975) Plunt Physiol. 55, 2-29; Kul, K., nd Lester, R. L. (978) Biochemistry 7, ) with the simplest nd most bundnt components hving the structure GlcN(kAc)(l + 4)GlcUA(l + 2)myoinositol-l-Ophosphorylcermide (Hsieh, T. C.-Y., Kul, K., Line, R. A., nd Lester, R. L. (978) Biochemistry 7, ). To determine the structure of the more complex members of this series, mixture of oligoscchrides ws prepred from crboxyl-reduced glycophosphocermide concentrte by lkli-ctlyzed hydrolysis nd lkline phosphtse tretment. A combintion of reverse-phse high pressure liquid chromtogrphy (Wells, G. B., nd Lester, R. L. (979) Anl. Biochem 97, 84-9), norml-phse high pressure liquid chrom- sulting intct oligoscchrides prior to norml nd reversedphse chromtogrphy. Frctions were further purified by togrphy, nd thin lyer chromtogrphy were used to thin lyer chromtogrphy to yield triscchride nd two resolve severl oligoscchrides s cetylted deriv- tetrscchride frctions in pure form. tives. Products of methyltion nlysis, Cr3 oxidtion, EXPERMENTAL PROCEDURES nd decetyltion-demintion were identified using chemicl ioniztion mss spectrometry to give the fol- A glycophosphocermide concentrte from tobcco leves ws lowing novel structures. A mjor tetrscchride ws prepred by Kul nd Lester (6) with molr rtio of hexuronic cid completely chrcterized s Gl( + 4)GlcNAc(l + gucose:phosphorus of (.5:.4:.). Hexuronic cid ws ssyed c- 4)GlcUA(l + 2)myoinositol. An dditionl structure cording to Blumenkrntz nd Asboe-Hnsen (7). Glucose ws determined by gs-liquid chromtogrphy of glucitol cette prepred fter of minor tetrscchride ws prtilly chrcterized s GlcNAc(cx -+ 4)GlcUA(l -+?)myoinositol(? + )Mn. These re representtives of clss of cidic glycolipids from plnts, possibly nlogous to the cidic gngliosides found in niml cell membrnes. Glycophosphocermides, s inositol-contining cidic glycolipids, occur widely in plnts nd fungi (-3), but few exmples of perhps 2 or more existing compounds hve been reported s chemiclly chrcterized (4, 5). This hs been due to the extreme difficulty in isoltion of chemiclly nlyzble mounts of individul members from glycophosphocermide mixtures. Pioneering work in this re ws performed by Crter et l. (, 4). Recently, we reported the * A preliminry report of this work ws presented t the Americn Chemicl Society meeting in Wshington, D. C.,979 ((98) Am. Chem. SOC. Symp. Ser. 8, 65-78). The costs of publiction of this rticle were defryed in prt by the pyment of pge chrges. This rticle must therefore be hereby mrked dvertisement in ccordnce with 8 U.S.C. Section 734 solely to indicte this fct. $ Present ddress, R nd D, Brown nd Willimson Tobcco Corp., 6 West Hill St., Louisville, KY To whom correspondence should be ddressed. Recipient of Reserch Grnt PCM76934 from the Ntionl Science Foundtion, Project KTRB53 from the Tobcco nd Helth Reserch nstitute, University of Kentucky. Recipient of Grnt GM2392 from the Ntionl nstitutes of Helth complete structure of cermide-phosphotriscchride which comprises 2% of the glycophosphocermide concentrte prepred from tobcco leves (5). This structure, GlcNAc(cul4 4)GlcUA(l + 2)inositol---phosphorylcermide, my be bsic core structure of the tobcco glycophosphocermides. More complex glycolipids in the concentrte were shown to contin glctose nd rbinose in ddition to the triscchride constituents (5). We now describe the preprtion of n oligoscchride concentrte from which we hve prepred dequte quntities to chrcterize completely the mjor tetrscchride phosphocermide. The concentrte ws obtined by fist removing the phosphocermide component from the crboxyl-reduced glycophosphocermide mixture nd then cetylting the re- hydrolysis in 2 N trifluorocetic cid t 2 C for 9 min (8). Phosphorus ws determined fter perchloric cid digestion ccording to Brtlett (9). The myoinositol-contining discchride glctin (~-L--O-CY-Dglctopyrnosylmyoinositol) ws gift from Dr. Clinton Bllou, University of Cliforni, Berkeley. Unless otherwise indicted ll high pressure liquid chromtogrphy ws crried out with pir of Model 6A pumps controlled by Model 66 progrmmer (Wters Assocites, Milford, MA) nd detection ws performed with moving-wire flme-ioniztion detector (Model LCMP, Pye Unicm Ltd., Cmbridge, Englnd). N-Acetyltion-About 5% of the glycophosphocermide from tobcco leves contin glucosmine without the N-cetyl group (3, 6). N-Acetyltion ws crried out ccording to Rosemn nd Dffner () on 5 g of the glycophosphocermide concentrte. Since N-cetylted hexosmines re more esily liberted by cid hydrolysis thn the uncetylted hexosmine (ll), the rtio (hexosmine hydrolyzed in 2 N HC t C for khexosmine hydrolyzed in 6 N HCl t C for 8 h) ws used to monitor the extent of the N-cetyltion of the glycophosphosphingolipid concentrte. The colorimetric procedure for hexosmine fter hydrolysis ws ccording to Gtt nd Bermn (2). The rtio of 2 N HC-relesble hexosmine doubled fter one ddition of cetic nhydride nd remined the sme fter second ddition. Therefore, the N-cetyltion ws considered essentilly complete. The N-cetylted concentrte ws lyophilized nd resuspended in 66 ml of wter nd pssed through 2-ml column of ction exchnge resin (AG 5W-X8, H form, 2-4 mesh, Bio-Rd). A totl of 2 ml of elute in wter ws collected nd lyophilized. Crboxyl Reduction to Convert Glucuronic Acid to Glucose in the Glycophosphocermide Concentrte-The crboxyl-reduction procedure ws dpted from tht of Tylor et l. (3), yielding product

2 7748 Glycophosphocermides Leves Tobcco from with uronic cidgucose:phosphorus rtio of..3.. The smple ws then dilyzed nd lyophilized. Recovery bsed on phosphorous ws 94%. Removl ofphosphocermide-an liquot of the crboxyl-reduced glycophosphocermide concentrte contining.4 mmol of totl phosphte ws dissolved in 6 ml of.5 N KOH nd heted t "C for 8 h. The smple ws cooled to room temperture, 2.8 ml of glcil cetic cid were dded, nd the ph ws djusted to 3 with 2 ml of 4 N HC. A totl of 6 ml of chloroform ws used to extrct lipids from the smple. The queous lyers were combined nd ph ws djusted to 8 with 6 mlof5 N NH4H nd the remining chloroform ws removed by rotry evportion. The ph ws redjusted to 8 nd the white precipitte formed ws removed by centrifugtion. The mixture of oligoscchride nd phospholigoscchrides obtined t this stge ws treted with severl 2 unit doses of Escherichi coli lkline phosphtse (Sigm P-4377) to remove the phosphte. After 56 h, 82% of totl phosphte ws relesed s P,. The smple ws pplied to 2-ml column of Dowex -X2 resin (bicrbonte form, 2-4 mesh) to isolte the dephosphorylted oligoscchrides free of phosphte nd phosphorylted impurities. The yield t this step, bsed on pentose, ws 76%. Since the lkline hydrolysis lso N-decylted the glucosmine, the procedure of Rosemn nd Dffner () ws gin used to N-cetylte the oligoscchride mixture. The smple ws then pssed through 2-ml column of AG 5W-X8 (H' form, 2-4 mesh). Unbound frctions were collected in 3 column volumes of wter. Rotry evportion t 35 "C gve syrup-like smple. Absolute ethnol ws dded in 2-ml quntities six times during further rotry evportion to remove wter. Five mlof redistilled pyridine/cetic nhydride (:l) were dded to the oligoscchride mixture. Percetyltion ws crried out t C for.5 h. Redistilled toluene ws used to remove cetic nhydride s n zeotrope t 4 "C under nitrogen. The percetylted oligoscchride mixture ws extrcted with 5 ml of redistilled chloroform three times ginst 5 ml of wter. The chloroform lyers were combined. Hi-FLosil Liquid Chromtogrphy of the Percetylted Oligosc- chride Mixture-The percetylted oligoscchride mixture ws chromtogrphed on column (2.54 X cm) of silic gel (Hi-Flosil, 6/2 mesh, Applied Sciences, Ltd.). n ech pilot run, smple contining 2.4 pmol of hexosmine ws pplied to the column. Specific solvent compositions re described in the legend for Fig.. n preprtive run, smple contining bout 3 pmol of hexosmine ws pplied to the sme column nd chromtogrphed under similr conditions. Grdient Reverse-phse High Pressure Liquid Chromtogrphy of Hi-Flosil Frction 3 from Fig. -The reverse-phse HPLC' conditions used to frctionte the percetylted oligoscchride mixture were dpted from those of Wells nd Lester (4). n ech nlyticl run, n liquot of Hi-Flosil Frction 3 contining 2.5 pmol of hexosmine ws dissolved in 5 pof cetonitrile nd chromtogrphed on n octdecyl reverse-phse column (.46 X 25 cm, Prtisil 5-ODS, Whtmn) (Fig. 2). n preprtive runs 3 pmol of Frction 3 were chromtogrphed on column (.45 X 2 cm) of CS-Corsil (37/5 pm, Wters Assocites) kept t 65 "C (Fig. 3). Frction C of Fig. 2 is comprble to Frction 4 of Fig. 3. socrtic Reverse-phse High Pressure Liquid Chromtogrphy of Oligoscchride Frction 5 from Grdient HPLC in Fig. 3- Oligoscchride Frction 5 (Fig. 3) from the reverse-phse HPLC grdient chromtogrphy ws chromtogrphed on the Ci-Corsil reverse-phse column isocrticlly in cetonitrile wter (3:7)(see Fig. 4). Thin Lyer Chromtogrphy-Silic Gel G pltes with 25pm thickness or high efficiency Silic Gel G pltes (HETLC, Anltech, nc.) were used. For cetylted sugrs the TLC solvent ws chloroform/methnol rnging in composition from 5:l to 7:l. For uncetylted sugrs, n cetonitrile wter solvent system in proportions of 3:l to 5:l ws used. Sugr spots were visulized by sprying the plte first with solution of orcinol (2 mg of orcino/ ml of 95% ethnol in wter) nd then with 75% sulfuric cid in wter followed by heting t "C for 5 to min (Fig. 5). Norml-phse High Pressure Liquid Chromtogrphy-Further seprtion of the mjor components in the grdient HPLC oligoscchride Frction 5 (Fig. 3) ws chieved by norml-phse high pres- ' The bbrevitions used re: HPLC, high pressure liquid chromtogrphy; nos, myoinositol; GC/MS, gs chromtogrphy/mss spectrometry; C, chemicl ioniztion; E, electron impct; TLC, thin lyer chromtogrphy. PSL nd, glycophosphosphingolipids nd. sure liquid chromtogrphy on column (.39 X 3 cm) of pporsi (Wters Assocites) eluted isocrticlly with chloroform:2-propnol (98.5.5). The flow rte ws ml/min (Fig. 6). Thin lyer chromtogrphy from frctions of this run is shown in Figs. 7 nd 8. Determintion of Crbohydrte Composition nd Anomeric Con- figurtion of Tetrscchrides A nd B-To n liquot contining 4 nmol of the mjor tetrscchride (B) (Frctions 9-7 in Fig. 6) or the minor tetrscchride (A) (Frctions 5-7 in Fig. 6) ws dded 5 nmol of xylitol (s internl stndrd). The smple ws dried under nitrogen nd cetylted in ml of pyridine/cetic nhydride (:, v/ V) t "C for h. One-hlf of the smple ws then evported to dryness, mixed with.5 ml of glcil cetic cid nd 5 mg of CrO:3, nd kept for 5-2 min t 4 "C in n ultrsonic bth. The mixture ws then diluted with 3 mlof wter nd extrcted with 2 mlof chloroform three times. The combined chloroform lyers were extrcted with.5 ml of wter before evportion to dryness. The bove procedures were dpted from those of Hoffmn et l. (5) nd Line nd Renkonen (6). The smple ws then hydrolyzed t 2 "C for 9 min in.5 ml of 2 N trifluorocetic cid. After evportion to dryness under nitrogen, the smple ws reduced in.5mlof N NH,OH contining 2 mg of NBH4 t room temperture for 7 h. The smple ws cidified with glcil cetic cid to destroy NBHs nd borte ws removed by codistilltion with methnol repeted five times (7). After incubtion t "C for h in ml of pyridine/cetic nhydride (l:l), the smple ws evported to dryness severl times with ddition of.5 ml of redistilled toluene ech time. After ddition of 2 ml of wter, the mixture ws extrcted with 2 mlof chloroform four times. The chloroform lyers were then combined nd evported under nitrogen to dryness. The lditol nd myoinositol cettes thus obtined were dissolved in 25-5 pl of redistilled cetone nd nlyzed by GC s described below. The other hlf of the cetylted oligoscchride ws processed by the procedure described bove except tht CrOs ws omitted. This smple ws used to determine the crbohydrte composition which, on comprison with the composition obtined fter tretment, would give evidence for the nomeric configurtion of the glycosidic linkges. The lditol nd myoinositol cettes were chromtogrphed on glss column (5 cm X 2 mm inside dimeter) pcked with 3% OV- 275 on /2 Chromosorb WAW (Supelco, nc.) with nitrogen s crrier gs t flow rte of 4 ml/min in Perkin-Elmer gs chromtogrph (Model 392) equipped with flme-ioniztion detector. Column temperture ws progrmmed from 2-25 "C t liner rte of 4 "C/min. Pek re ws clculted by tringultion. Methyltion Anlyses of Oligoscchrides by GC/MS-Aliquots from the oligoscchride frctions t vrious stges of isoltion were tken to prepre permethylted oligoscchrides using methylsulfinyl-crbnion (8) nd were subsequently derivtized to prtilly methylted lditol nd myoinositol cettes ccording to Bjorndl et l. (9, 2) nd Stellner et l. (2) with deuterium lbeling t the C- position of the lditols by use of sodium borodeuteride. The cettes were chromtogrphed nd nlyzed in Finnign gs chromto- grph/mss spectrometer/dt system (Model 33/6), using both electron impct nd chemicl ioniztion mss spectrometry. Detiled E or C GC/MS conditions re described in the figure legends. Crbohydrte Sequence Determintion of the Mjor Tetrscchride (Decetyltion-Demintion)-Approximtely 6 nmol of percetylted mjor tetrscchride (Frctions 9-7 in Fig. 7) ws - nd N-decetylted by heting t "C for 2 h in. ml of.5 N KOH in wter. The ph ws djusted to 6 with glcil cetic cid. The mixture ws then deslted on column (5 X.4 cm) of Bio-Gel P- 2 (Bio-Rd) in 5% cetic cid. A smll liquot from ech frction ws spotted on Silic Gel G thin lyer plte nd the sugr ws visulizedby orcinol/h3o4 spry. Orcinol-positive frctions were pooled nd evported to dryness under reduced pressure. For demintion, 2. ml of the nitrous cid regent, prepred ccording to Byrd nd Roux (22), were dded to the smple. After 8 h t room temperture, the demintion mixture ws pplied to -ml column of AG 5W-X4 (H' form, 5- mesh, Bio-Rd) nd eluted with 35 ml of wter. The elute ws evported to dryness under reduced pressure, tken up in ml of wter, pplied to -ml column of AG -X8 (cette form, 2-4 mesh, Bio-Rd), eluted with 35 ml of wter, nd evported to dryness. One-hlf ml of N NH4OH contining 5 mg of NBD4 ws used t room temperture for 6 h to reduce the demintion products. After cidifiction with glcil cetic cid to destroy NBD4, the smple ws evported with methnol t 4 "C under nitrogen to remove borte. The mixture ws pssed through both ction nd nion exchnge resins gin s described bove. The elute ws evported to dryness nd tken up

3 Glycophosphocermides from Tobcco Leves 7749 n in.5 ml. An liquot ws dried nd permethylted using methylsulfinyl-crbnion (8) nd subjected to C GC/MS nlysis. For detiled GC/MS conditions see legend for Fig.. RESULTS.w Hi-Flosil Liquid Chromtogrphy of the Percetylted - A - Oligoscchride Mixture-The percetylted oligoscchride mixture obtined from the crboxyl-reduced glycophos phosphingolipid concentrte ws chromtogrphed in Hi- Flosil silic gel column s shown in Fig.. The mjor frction, c Frction 3 (Fig. l), contined mixture of oligoscchrides - w further resolved by reverse-phse HPLC s described below. Higher number frctions (Frctions 4-) contined higher W oligomer oligoscchrides nd remin to be investigted. Anlyticl Reverse-phse High Performnce Liquid Chro- M mtogrphy of the Mjor Hi-Flosil Frction (Frction 3) on Prtisil-ODs-Hi-Flosil Frction 3 (Fig. ) ws chromtogrphed on n nlyticl reverse-phse column s shown in Fig. 2. The mjor frction (Frction C) in Fig. 2 hs the sme w n ELUTON VOLUME (ml FG. 3. Preprtive reverse-phse chromtogrphy of the mjor Hi-Flosil frction (Frction 3 in Fig. ) of percetylted oligoscchrides. A nonliner solvent grdient from cetonitrile/ wter (:9-6:4) ws used for elution. Flow rte ws 2 ml/min. For other chromtogrphic conditions, see Experimentl Procedures. Frctions pooled re indicted by numbers under the peks ELUTON VOLUME (ml) FG.. Hi-Flosil liquid chromtogrphy of percetylted oligoscchride mixture. Solvent A is chloroform/methnol (6:l). Solvent B is chloroform/methnol (:l). Soluent C is chloroform/ methnol/wter (6:6:5). Flow rte ws 9.9 ml/min. Other detils re described under Experimentl Procedures. Frctions pooled re indicted by verticl lines nd numbers under the chromtogrm. The mjor frction (Frction 3) ws utilized for further nlysis by reverse-phse high pressure liquid chromtogrphy. e ELUTON VOLUME (ml) FG. 2. Anlyticl reverse-phse high pressure liquid chromtogrm of themjor Hi-Flosil frction on Prtisil-ODS (Whtmn). A nonliner solvent grdient ws used s indicted by from cetonitrile/wter (:9-7:3) t flow rte of ml/min. Pooled frctions re indicted by verticl lines below the trcing nd lbeled A-Q. TABLE Hi-Flosil chromtogrphy yields Frction Weight (me) elution volume s the crboxyl-reduced nd cetylted triscchride derived from PSL (5): GlcNAcp(d+ 4)Glcp(l-+ 2)myoinositol. The lter peks, D-Q (Fig. 2), re higher oligomers contining glctose nd/or rbinose. Methyltion Linkge Anlyses of Prtisil-ODS Frctions from Fig. 2-For preliminry knowledge bout the kinds of glycosidic linkges present in nlyticl frctions from the Prtisil-ODS chromtogrphy, methyltion nlyses were crried out. The mjor frction, Frction C in Fig. 2, hd the sme linkge type s those of the PSL crboxyl-reduced triscchride, terminl N-cetylglucosmine, 4-linked glucose, nd monosubstituted myoinositol (5), nd is therefore considered to hve the sme structure s indicted bove. Further chrcteriztion of the frction s the PSL crboxyl-reduced triscchride will be discussed below. Other frctions (Frctions D-Q, Fig. 2) hd substitutions on the scchrides indicting the presence of terminl rbinose, terminl glctose, 3,6-brnched glctose, 4-linked N-cetylglucosmine, nd combintions of these. These methyltion dt s well s results from thin lyer chromtogrphy (not shown) indicted tht the most bundnt oligoscchride in the mixture (Frction C in Fig. 2) ws triscchride which most likely rose from the mjor glycophosphosphingolipids (PSL nd PSL ) in the concentrte. Preprtive Reverse-phse Grdient High Pressure Liquid Chromtogrphy of the Mjor Hi-Flosil Frction-Hi- Flosil Frction 3 (Fig. ) ws chromtogrphed on preprtive reverse-phse column s shown in Fig. 3. Clusters of peks were nlogous to those oserved in nlyticl runs on the Prtisil-ODS column (Fig. Z), except tht resolution ws somewht scrificed within ech cluster in these preprtive runs. Frctions were pooled nd evported to dryness under reduced pressure. The weight of ech frction (s percetylted derivtives) is shown in Tble. These numbers represented the yield of ech frction derived from totl of 3.5 g of the glycophosphosphingolipid crude concentrte used to prepre this btch of the crboxyl-reduced oligoscchride mixture. t is not excluded tht smll mounts of noncrbohydrte impurities could still be present in these frctions.

4 Glycophosphocermides Leves Tobccofrom 775 Preprtive Zsocrtic Reverse-phse High Pressure Liquid chromtogrphed on TLC in chloroform/methnol (4:l) s shown in Fig. 7. Frctions 3-7 of the pporsil HPLC (Fig. 6) Chromtogrphy of the Tetrscchride Frction-Pooled Frction 5 (Fig. 3, tetrscchride frction) from the grdient contined single spot on TLC (tetrscchride A) which preprtive reverse-phse Cln-Corsilcolumn ws chromto- migrted with n RFless thn thtof the PSL triscchride. grphed gin on the sme column isocrticlly with ceto- The mjor oligoscchride component (tetrscchride B), nitrile/wter (3:7) further to resolve the individul oligosc- which distributed minly in Frctions 8-7 of the pporsil chrides s shown in Fig. 4; Frctions 3 nd 4 represented chromtogrm (Fig. 6), hd n RF less thn tht of tetrscmteril eluted with %cetonitrile. chride A. Pool X mteril from the isocrtic reverse-phse HPLC Pooled frctions from isocrtic reverse-phse chromtogrphy (Fig. 4) were dried underreduced pressure nd liquots (Frctions 6-8, Fig. 4) wslso chromtogrphedonthe were nlyzed by TLC in chloroform/methnol (4:), s pporsil column under identicl conditions. The chromtoshown in Fig. 5. Frctions 5-8 from theisocrtic reverse-phse grm (not shown) wssimilr to Fig. 5 except tht the minor step (Fig. 4) showed only single spots. n Frctions 9-2 from pek (tetrscchride A)ws not present. Pooling of Oligoscchrides A nd B from Clporsil Liquid Fig. 4, the mjor component seen on TLC (chrcterized s tetrscchride B nd discussed below) ws well seprted Chromtogrphy-The pporsil frctions corresponding to (Frctions 5 nd 6, Fig. 5) fromseverl from the minor component, tetrscchride A, which hd theminorpek higher RF (prtilly chrcterized s the minor tetrscch- repetitive runs were combined. The frctions contined one ride nd discussed below). Since the chromtogrphyprinci- of the minor tetrscchrides, referred to s tetrscchride ples involved in silic gel TLC under the conditionsused re A. The pporsil frctions corresponding to the mjor pek similr to thoseof norml-phse HPLC onsilic gel columns, the ltterws used to seprte thesetwo oligoscchrides. As (Frctions 8-, Fig. 5) were combined. The mjor composhown in Fig. 4, Frctions 6-8 were combined (Pool X) nd nent, referred to s tetrscchride B, is the mjor tetrscchride isoltedfrom the crboxyl-reducedglycophosphosfrctions 9-2 were combined (Pool Y). Norml-phse High Pressure Liquid Chromtogrphy of phingolipid concentrte of tobcco leves. Aliquots of the purified tetrscchrides were chromtothe Tetrscchride Frction-Pool X nd Pool Y (Fig. 4) were chromtogrphed on pporsi (Wters Assocites) with chloroform:2-propnol (98.5:.5). Fig. 6 showsthenormlphse HPLC chromtogrm of Pool Y. The pooled frctions were evported to dryness under nitrogen nd tken up in pl of chloroform. An liquotfromechfrction ws - -- MNOR TETRASACCHARDE w h8. -MAJOR i TETRASACCHARDE t T A ELUllON VOLUME (ml) FG.4. socrtic reverse-phse (C8-Corsil)liquid chromtogrm of pooled Frction 5 from preprtive reverse-phse liquid chromtogrphy (Fig. 3). The column ws eluted with cetonitrile/wter (3:7) isocrticlly until Frction 2 ws obtined (5ml). Frctions 3 nd 4 were eluted with %cetonitrile. The flow rte ws 2 ml/min. For chromtogrphy detils, see Experimentl Procedures. Frctions pooledwere indicted by numbers below the chromtogrm. Frctions6-8 were combined into Pool X. Frctions 9-2 were combined into Pool Y. FG.5. Thin lyer chromtogrmof frctions from isocrtic reverse-phse liquid chromtogrphy. An liquot from ech pooled frction ws spotted nd lbeled by numbers from through 4 (see Fig. 4). Lne T ws n liquot of the crboxy-reduced nd percetylted PSL triscchride. Lne A ws n liquotof the totl smpleppliedtothe column. Therrowpointstothe origin. Chloroform/methnol ( 4 ) ws used s developing solvent. Sugr spots were visulized by orcinol/hb4 spry s described under Experimentl Procedures. Grdul increse in RF vlues observed in smples chromtogrphed on lnes towrd both sides of the TLC plte ws due to solventedge effect. FG. 6. Norml-phse liquid chromtogrm of thetetrscchride frction (Pool Y,Fig. 4) on pporsil column. Frctions pooled re indicted W m z B m W by verticl lines below the chromtogrm nd numbers bove. Chromtogrphy of Pool X under identicl conditions gve similr brod pek except the minor pek (Frctions 5-7) ws bsent(notshown). See Experimentl Procedures for detils. Q: Q: E! U W W ELUTON VOLUME (ml)

5 Glycophosphocermidesfrom Tobcco Leves T A 8 9 O FG. 7. Thin lyer chromtogrm of pporsil frctions of Pool Y (see Fig. 5). An liquot of p out of p of ech pporsil frctionws spotted on silic gel plte. Numbers correspond to frction numbers from the yporsil column (Fig. 6). Lne 2. n liquot of crboxyl-reduced nd percetylted triscchride of PSL. Lne A, n liquot of Frction 5 from Cls-Corsil chromtogrphy (Fig. 3). Therrow indictes the origin. Chloroform/methnol (4) ws used for development nd the sugr spots were visulized by orcinol/h2s4 spry. M NOERT R A S A C C H A R D f m e- J C i MAJOR flrasaccharde FG. 8. Thin lyer chromtogrm of pooled cetylted tetrscchride frctions on high efficiency silic gel (HETLC, Anltech, nc.). Origin is mrked by rrow. The plte ws developed in chloroform/methnol (35:l) twice. Sugr spots were visulized by orcinol/h2s4 spry. Lne, PSL triscchride; Lne 2. Frction 5 from Clx-Corsil (Fig. 3) chromtogrphy; Lne 3, tetrscchride A Lnes 4-6, tetrscchride B. 775 gs chromtogrphy. Molr quntities of ech sugr were clculted per mol of myoinositol. Tble shows the dt with the molr rtio nd nomeric configurtions ssigned. The composition dt suggest tht theminor tetrscchride A contins equimolr mounts of Mn, Glc, GlcNAc, nd nos, while the mjor tetrscchride B contins equimolr mounts of Gl, Glc, GlcNAc, nd nos. The ssumption tht these components re tetrscchrides rests on their retention on reversedphse column(4) nd on the fct tht glycosidic ssembly of the four components cn only led to tetrscchride. According to the studies on Angyl nd Jmes (23), Hoffmn et l. (5), nd Line nd Renkonen (6), tretment of percetylted hexopyrnosides with CrO in cetic cid, in which the glyconeoccupies nequtoril position, gives cetylted 5-hexulosontes. The corresponding nomers in which the glycone occupies n xil position re oxidized only slowly. Thus, thep-glycosidic D-hexopyrnoside units of the crbohydrte chin should be oxidized, wheres -glycosidic units should not. The corresponding - nd P-furnosides re redily oxidized, yielding 4-hexulosontes. As shown in Tble, ll the sugrresidues in tetrscchride A survivedcros tretment nd,hence, llthree glycosidic linkges were ssignedthe configurtion. n tetrscchride B, both Glc nd GlcNAc survived more thn 8% nd were lso ssigned the configurtion, wheres the glctosidic linkge ws ssigned P configurtion since only 29%of Gl survived the CrOs test. The ltter vlue ws little higher thn expected forn idellyselectiveoxidtion;however, Line nd Renkonen (6) observed similr resistnce of Pglctose units. Methyltion Linkge Anlysisof the Mjor Tetrscchride-Tetrscchride B (Frction 4, Figs. 6 nd 7) ws processed for linkgenlysis s described under Experimentl Procedures. Fig. 9 shows the totl ion chromtogrms obtinedunder identicle GC/MS conditions from the prtilly methylted lditol cettes of the following smples: Z, pporsil Frction 4 (Fig. 6) (mjor tetrscchride B); ZZ, the crboxyl-reduced triscchride of PSL (GlNAc( + 4)Glc(l+ 2)inositol); ZZZ, lctose; nd ZV, glctin (Gl( + )inositol). E mss spectr from the four mjor peks in the gs chromtogrm represented by Fig. 9 were identicl with those from uthentic stndrds (not shown). Pek C (Fig. 9) ws ssigned s 2,3,4,6-tetr-O-methyl-,5-di-O-cetylglctitol from its mss spectrum. Thisindictes the presence of terminl glctose in the mjor tetrscchride. TABLE grphed on high efficiency silic gel thinlyerplte in Determintion of crbohydrte compositions nd nomeric chloroform/methnol(35:).the chromtogrm visulized by configurtions of tetrscchrides A nd B orcinol/h2s4 spry is shown in Fig. 8. The minor tetrscmoles per mol of myoinositol detected chride, A, migrted s single bnd in Lne 3 (Fig. 8). The mjortetrscchride, B, fromvrious pooled frctions is Mn Glc Gl GlcNAc lnos shown in Lnes 4-6 (Fig. 8). The RF of the PSL crboxyl- Tetrscchride A reduced nd percetylted triscchride (Lne ) is higher Before CrO:, thn therfof percetylted tetrscchridea (Lne 3), both After CrO:, 6% 97% being higher thn thtof tetrscchride B (Lnes 4-6) (see % survivl 7 % Fig. 8). The TLC suggests tht tetrscchride B is the mjor Molr composition component in Frction 5 from thec8-corsilchromtogrhy Anomeric configur (Fig. 8, Lne 2) while tetrscchride A occurs in smller tion mounts in the sme C8-Corsilfrctions. Net weights (percetylted) of these pooled frctions fter solvent evportion Tetrscchride B Before CrOil were s follows: 4.8 mg of minor tetrscchride A, 8.5 mg of After CrO.,.6. mjor tetrscchride B. 8% 29% % survivl 88% % Determintion of Crbohydrte Composition n d Ano Molr composition meric Configurtion of Tetrscchrides A n d B-Alditol P nd myoinositol cettes, prepred from the tetrscchrides Anomeric configurtion A nd B before nd fter CrO:l oxidtion, were nlyzed by

6 7752 Glycophosphocermides from Leves Tobcco myoinositol in the PSL triscchride s chrcterized by periodte oxidtion experiments (5). The --cetyl isomer from glctinol hd different retention time of.9 min under these conditions, shown s Pek B in Fig. 9ZV. These methyltion nlyses indicted tht the oligoscchride in pporsil Frction 4 (Fig. 6, the mjor tetrscchride, or tetrscchride B) contined the following types of sugr linkges: terminl Gl, 4-linked GlcNAc, 4-linked Glc, nd 2- linked myoinositol. n seprte experiment prtilly methylted lditol cette smple prepred from the grdient Prtisil-ODS Frction C (Fig. 2) contined triscchride which ws the most bundnt oligoscchride in the oligoscchride concentrte (Fig. 9). This result indicted tht the mjor triscchride (Prtisil ODs, Frction D) shred the sme structure s the PSL crboxyl-reduced triscchride GlcNAcp( + 4)Glcp(l+ 2)nos, s lredy reported (5). Methyltion Linkge Anlyses of the Mjor Tetrscchride nd the Minor Tetrscchride by C Mss Frgmentogrphy-To verify the linkge nlyses of tetrscchride B (pporsil Frction 4) nd to obtin linkge informtion on the smll mount of tetrscchride A isolted, more sensitive pproch, chemicl ioniztion mss frgmentogrphy, ws used to nlyze the prtilly methylted lditol cettes prepred from tetrscchrides A nd B. The distribution of the ion frgment t m/z 2 (MH-cetic cid-methnol, M = 292) which is chrcteristic ion for SPECTRUM NUMBER FG. 9. Totl ion chromtogrms of prtilly methylted lditol cettes of: Z, pporsil Frction 24 (Fig. 6); ZZ, PSL crboxyl-reduced triscchride; ZZZ, lctose; ZV, glctinol. Ech of the prtilly methylted lditol cettes mixture ws chromtogrphed on glss column (5 cm X 2 mm inside dimeter) pckedwith 3% OV-2 (Supelco,nc.). Flow rte of the helium crrier gs ws 3 min/min. Column temperture ws progrmmed from 5-2 "C t 6 "C/min. Seprtor nd trnsfer line tempertures were 22 "C. MS nlyzer temperture ws 6 "C. Electronimpctionsource prmeters were: lens, 2 V, collector, 35 V; extrctor, 2.7 V; ion energy, 2.5 ev (progrmmed); electron energy, 7 ev; ion volume,.54 ma; emission current,.6 ma. Mss spectrl scns of GC effluent were recorded t rte of 2 s/spectrum from m/ z 4 to m/z 25. GC retention time in seconds cn be clculted by multiplying the spectrum number (bsciss) by Pek C in Fig. 9 hd the sme GC retention time of 6.4 min - nd the sme mss spectrum s those of non-reducing terminl glctose (Pek C) from lctose in Fig. 9, nd terminl Gl z5-nh-mn glctose (Pek C) from glctinol (Fig. 9V). Pek D in Fig. 9 ws ssigned s 2,5,6-tri-O-methyl-2,4,5- NBD4 tri--cetyl-glucitol indicting the presence of 4-linked glu- Reduction WS cose in the tetrscchride from pporsil Frction 4 (Fig. 6). This pek (Fig. 9) hd the sme GC retention time of 9. min s those of Pek D from the PSL crboxyl-reduced triscchride (4-linked glucose) (Fig. 9) nd Pek D from lctose (4-linked glucose) (Fig, 9). n ddition, the E Permethyltion spectrum showed fetures chrcteristic for the ssignment of c $' 4-linked glucose. Pek Fin Fig. 9, identified s 3,6-di-O-methyl-l,4,5-tri-Ocetyl-2-deoxy-N-methylcetmidoglucitol, rises from 4- linked GlcNAc in the tetrscchride of pporsil Frction 4 (Fig. 6). Pek A in Fig. 9 ws postulted to be,3,4,5,6-pent-omethyl-2--cetylmyoinositol derived from 2-linked myoinositol. This pek hd the sme retention time (.2 min) s FG.. Scheme for demintive clevge of tetrscch- Pek A in Fig. 9, which ws derived from the 2-linked ride B, followed by reduction of the products.

7 Glycophosphocermides from Tobcco Leves 7753 pent-o-methyl-mono-o-cetylmyoinositol gve gs-chromtogrphic pek (not shown) derived from monosubstituted myoinositol in the tetrscchride (5). Neutrlosses of cetic cid nd methnol from the ions re common in frgmenttion under the methne C MS conditions used. Another pek showed the distribution of n ion t m/z 264 (MHcetic cid, M = 323) which is chrcteristic for tetr-- methyl-di--cetyl--deutero-glctitol derived from terminl Gl in the tetrscchride. A third pek showed the profie of ions t m/z 292 (MH-cetic cid, M = 35) which is chrcteristic for tri--methyl-tri--cetyl--deutero-glucitol derived from 4-linked Glc in the tetrscchride. A finl pek showed the distribution of n ion t m/z 393 (MH, M = 392) which is chrcteristic for di-o-methyl-tri-o-cetyl-2- N-methyl-2-cetmido-2-deoxy-l-deutero-glucitol derived from 4-linked GlcNAc in the tetrscchride. The bove dt from C mss frgmentogrphy is consistent with the dt from E GC/MS for the mjor tetrscchride B. Scns of pproprite ions showed the bsence of brnching structures in oligoscchride B. Another C mss frgmentogrm of PMAA prepred from tetrscchride A gve pek showing the distribution of n ion t mjz 264 (MH-cetic cid, M = 323) which is chrcteristic ion for tetr--methyl-di--cetyl--deutero-mnnitol derived from terminl Mn. A second pek showed the distribution of n ion t m/z 292 (MH-cetic cid, M = 35) which is chrcteristic ion for tri-o-methyl-tri-o-cetyl-- deutero-glucitol in this cse derived from 4-linked glucose. Becuse of the terminl GlcNAc, 4-linked Glc, nd terminl Mn, brnched (or disubstituted) structure, ws proposed for the myoinositol residue in tetrscchride A. A well defined pek for the myoinositol derivtive ws not pprent becuse of the smll mount of mteril vilble. A prtil structure is proposed s follows for the minor tetrscchride: GlcNAcp( + 4)Glcp(l-+?)nos(? c )Mn. Crbohydrte Sequence Determintion of the Mjor Tetrscchride B-The sugr composition nd the methyltion dt suggest the following two possible sequences since the mono-substituted myoinositol (e.g. 2-linked myoinositol) must be t the "reducing end" of the scchride chin nd terminl glctose must be t the nonreducing end: Glp(bl+ 4)GlcNAcp(l+ 4)Glcp(l-+ 2)nos () Glp(P 4 4)Glcp((l -$ 4)GlcNAcp(l -j 2)nos. () Sequence ws proved to be correct for this mjor tetrscchride by nitrous cid demintion experiments. Demi- 2. t L w L c z SPECTRUM NUMBER FG.. Crbohydrte sequence determintion of the mjor tetrscchride by C GC/MS of demintion products. Totl ion chromtogrm of reduced nd permethylted products of demintive clevge of A'-decylted tetrscchride B. Pek is permethylted Glcp(- 2)inositol nd Pek is permethylted Glp( - 4)2,5-nhydromnnitol. GC/MS conditions: column, 5 X.2 cm pcked with 3% OV on Supelcoport l/2 with methne s crrier nd regent gs supplying Torr pressure in the ion source; column temperture, 9 "C; injector nd trnsfer lines, 225 OC; ionizing electron energy, 5 ev; ion source temperture, 6 "C; scn rte, 4.2 s/ complete spectrum. i M. 3 4th K i FG. 2. Methne chemicl ioniztion mss spectrum of Pek Zin Fig.. Conditions were s described in the legend to Fig.. SCHEME ntion of the glucosmine residue specificlly cleves the glucosminic linkge in the tetrscchride, which would give mixture of Gl( + 4)2,5-nhydromnnitol nd Glc( + 2)nos, if the sequence were Gl( + 4)GlcNAc(l + 4)Glc(l + 2)nos, or mixture of Gl( + 4)Glc(l 3 4)2,5-nhydromnnitol nd myoinositol, if the sequence were Gl( + 4)Glc(l+ 4)GlcNAc(l"+ 2)nos. The percetylted mjor tetrscchride ws - nd N- decylted by KOH tretment. After deslting, the smple ws deminted in cetic cid with nitrous cid. Since 2,5- nhydromnnitol is expected to be t the reducing end of one of the oligoscchride products, the smple ws reduced with NBD4 to give l-deutero-2,5-nhydromnnitol. The products were then permethylted by the method of Hkomori (8) nd exmined by methne C GC/MS. The scheme for this procedure is shown in Fig.. Fig. shows the totl ion gs chromtogrm of the reduced nd permethylted products from demintive clevge of the mjor tetrscchride B showing two mjor products: nd Z. Fig. 2 is the mss spectrum of Pek in Fig.. The expected frgmenttion pttern (Scheme ) of per- methylted Glcp( -+ 2)nos, gives n ssignment of ll the prominent ions observed in the spectrum. ons t m/z 29, 87, nd 55 re probbly frgments of the A ring (glucose), while ions t m/z 233 nd 2 re from the B ring (myoinositol). The moleculr weight is 468 nd both MH (m/z 469) nd M- (m/z 467) were observed. Seril losses of methnol from M-, M, nd MH gve ions t m/z 435,436,437,45, nd 373. The ion t m/z 293 is probbly frgment from the B ring

8 7754 Glycophosphocermides from Tobcco Leves 36 FG. 3. Methne chemicl ioniztion mss spectrum of Pek ZZ in Fig.. Permethylted -+ 4)2,5-nhydromnnito; instrumentl conditions re described in the legend to Fig.. i SCHEME 2 plus the C- from the A ring with n dditionl -methyl group gined from C-2 or C-3 of the A ring through rerrngement (24, 25). n comprison with the spectrum of permethylted glctin obtined under identicl conditions, ll the sme prominent ions were present. Observtion of ions t mlz 293 hve lso been reported in spectr of permethylted discchrides contining myoinositol by Nccrto, et l. (26) nd by Lennrtson, et l. (27). Fig. 3 shows the mss spectrum of Pek (Fig. ). The frgmenttion pttern in Scheme 2 shows permethylted Glp(l+ 4)2,5-nhydrornnnitol with deuterium t the C- position of 2,5-nhydromnnitol. All of the prominent ion frgments were interpreted s follows. ons t m/z 29, 87, nd 55 re from the A ring (glctose), while the ions t m/ z 9 nd 58 re from the B ring (l-deutero-2,5-nhydromnnitol). The moleculr weight is 425 nd both M nd M- (m/ z 424) were detected. The ion t m/z 454 corresponds to M + C2H5. Seril losses of methnol from M-, M, nd MHgve prominent ions t m/z 392,394,362, nd 33. The ion t m/z 25 is probbly formed (nlogous to tht of m/z 293 in Fig. 9) from the B ring plus the C- of the A ring with n dditionl -methyl group gined from C-2 or C-3 of the A ring through rerrngement (24, 25) nlogous to the rtionle for the glucosyl-myoinositol. n conclusion, the C GC/MS determintion of the two methylted discchrides, hexosyl-myoinositol nd hexosyl- 2,5-nhydromnnitol, s the mjor demintion degrdtion products indicted tht the crbohydrte sequence of tetrscchride B ws Glp(D -+ 4)GlcNAcp(l + 4)Glcp(l -+ 2)nos. DSCUSSON Judging from the closely relted structures of the triscchride portions of the mjor glycophosphocermides. PSL (GlcNAc:GlcUA:nos, :l:l) nd PSL (GlcNH2:GlcUA:nos, l:l:l), to those of the mjor tri- nd tetrscchrides (GlcNAc: Glc:nos, :l:l; Gl:GlcNAc:Glc:nos, :l:l:l) isolted from the crboxyl-reduced glycophosphocermide concentrte, s well s the constnt presence of glucosmine, glucose, nd myoinositol in ll the other HPLC frctions (Prtisil-ODS or Ci- Corsil frctions), the glycophosphocermide members in this concentrte probbly represent series of complex lipids contining core triscchride moiety, GlcNAcp (or GlcNH2p) ( + 4)GlcUAp(l -+ 2)nos, with dditionl rbinose, glctose, or mnnose ttched to the triscchride. As the complexity increses, there is concurrent decrese in bundnce of the higher oligomers. The very smll mount of glucose (hexuronic cid/glucose, :.3) present before crboxyl reduction indicted tht the glucose residues in the oligoscchrides obtined from the crboxyl-reduced concentrte cme from glucuronic cid-contining glycophosphocermide. An entire series of cidic glycolipids is thus present in tobcco leves, nlogous in complexity to the gngliosides of niml tissue. t should be noted tht the glucosmine nd glucuronic cid residues in PSL nd PSL, s well s the glucosmine nd glucose residues in the mjor tri- nd tetrscchrides isolted from the concentrte, hve nomeric configurtion. The reported structure of "phytoglycolipid' by Crter nd coworkers hd this feture lso (4). Thisitution is unlike tht found in niml glycosphingolipids where N-cetylglucosmine is lmost lwys in the p configurtion. High pressure liquid chromtogrphy plyed criticlly importnt role in chieving seprtion nd isoltion of the mjor members in this closely relted complex mixture. Both norml-phse nd reverse-phse columns were employed under vrious combintions of solvent conditions to seprte the percetylted oligoscchrides (4). This study hs demonstrted the ppliction of the combintion of HPLC nd GC/MS in isoltion nd chrcteriztion of closely relted oligoscchrides in complex mixture nd points to fesible route to the future exmintion of the more complex members in the plnt glycophosphocermide fmily. Thistructurl biochemistry shll hopefully ly ground work for future studies of the function of these ubiquitous plnt-derived glycolipids. REFERENCES. Crter, H. E., Johnson, P., nd Weber, E. J. (965) Annu. Reu. Biochem. 34, Lester, R. L., Smith, S. W., Wells, G. B., Rees, D. C., nd Angus, W. W. (974) J. Biol. Chem. 249, Kul, K., nd Lester, R. L. (975) Plnt Physiol. 55, Crter, H. E., Strobch, D. R., nd Hwthorne, J. N. (969) Biochemistry 8, Hsieh, T. C.-Y., Kul, K., Line, R. A., nd Lester, 2. L. (978) Biochemistry 7, Kul, K., nd Lester, R. L. (978) Biochemistry 7, Blumenkrntz, N., nd Asboe-Hnsen, G. (973) Anl. Biochem. 54, Albersheim, P., Nevins, D. J., English, P. D., nd Krr, A. (967) Crbohydr. Res. 5, Brtlett, G. R. (962) J. Biol. Chem. 234, Rosemn, S., nd Dffner,. (964) Anl. Chem. 28, Whet, R. W. (966) Methods Enzymol. 8,6-78

9 Glycophosphocermides from Leves Tobcco Gtt, R., nd Bermn, E. R. (966) Anl. Biochem. 5, Tylor, R. L., Shively, J. E., nd Conrd, H. E. (976) Methods Crbohydr. Chem. 7, Wells, G. B., nd Lester, R. L. (979) Anl. Biochem. 97, Hoffmn, J., Lindberg, B., nd Svensson, S. (972) Act Chem. Scnd. 26, Line, R. A., nd Renkonen,. (974) J. Lipid Res. 6, Swrdeker, J. S., Sloneker, J. H., nd Jenes, A. (965) Anl. Chem. 37, Hkomori, S. (964) J. Biochem. (Tokyo) 55, Bjorndl, H., Lindberg, B., nd Svensson, S. (967) Crbohydr. Res. 5, Bjorndl, H., Lindberg, B., Pilotti, A., nd Svensson, S. (97) Crbohydr. Res. 5, Stelner, K., Sito, H., nd Hkomori, S. (973) Arch. Biochem. Biophys. 55, Byrd, B., nd Roux, D. (975) FEBS Lett. 55, Angyl, S. J., nd Jmes, K. (97) Aust. J. Chem. 23, Kovcik, V., Buer, S., nd Rosik, J. (968) Crbohydr. Res. 8, Lonngren, J., nd Svensson, S. (974) Adu. Crbohydr. Chem. 29, Nccrto, W. F., Ry, R. E., nd Wells, W. W. (975) J. Biol. Chem. 25, Lennrtson, G., Lundbld, A., Lindberg, B., nd Lonngren, J. (976) Biochem. Biophys. Res. Commun. 69,92-926